THE JOURNAL OF EXPERIMENTAL ZOOLOGY 261:340-348 (1992)

Expansion of Surface Epithelium Provides the Major Extrinsic Force for Bending of the Neural Plate IGNACIO S. ALVAREZ AND GARY C . SCHOENWOLF Department of Anatomy, University of Utah, School of Medicine, Salt Lake City, Utah 84132 ABSTRACT Neurulation, formation of the neural tube, requires both intrinsic forces (i.e., those generated within the neural plate) and extrinsic forces (i.e., those generated outside the neural plate in adjacent tissues), but the precise origin of these forces is unclear. In this study, we addressed the question of which tissue produces the major extrinsic force driving bending of the neural plate. We have previously shown that 1) extrinsic forces are required €or bending and 2) such forces are generated lateral to the neural plate. Three tissues flank the neural plate prior to its bending: surface epithelium, mesoderm, and endoderm. In the present study, we removed two of these layers, namely, the endoderm and mesoderm, underlying and lateral to the neural plate; bending still occurred, often with complete formation of a neural tube, although the latter usually rotated toward the side of tissue depletion. These results suggest that the surface epithelium, the only tissue remaining after microsurgery, provides the major extrinsic force for bending of the neural plate and that the mesoderm (and perhaps endoderm) stablizes the neuraxis, maintaining its proper orientation and position on the midline.

Formation of the neural tube from the ectoderma1 neural plate requires forces originating both intrinsically and extrinsically t o the neuroepithelium (reviewedby Schoenwolf and Smith, 'goal. Such forces are likely generated by a limited repertoire of cell behaviors that are common to a variety of morphogenetic processes (reviewed by Schoenwolf, '91). Traditionally, change in neuroepithelial cell shape from columnlike to wedgelike has been considered to play a paramount role in bending of the neural plate. However, quantitative studies have revealed that few cells actually become wedge shaped during bending (Schroeder, '70; Brun and Garson, '83; Schoenwolf and Franks, '84;Moore et al., '871, and those that do are restricted mainly to localized areas called hinge points (reviewed by Schoenwolf and Smith, '90b). Removal at the stage of the flat neural plate of the median hinge point, the area in which neuroepithelial cell wedging is concentrated during elevation of the neural folds, fails t o inhibit subsequent bending (Smith and Schoenwolf, '91>,but removal of tissue lateral t o the flat neural plate (i.e., the surface epithelium, mesoderm, and endoderm) blocks this process (Schoenwolf,'88) an exception occurs at the forebrain level, where bending is autonomous to the neural plate. Separation of the flat neural plate caudally from Hensen's node, a structure that regresses and, consequently, could stretch the neural plate (thereby aiding in its bending) has virtually no effect on bending (Schoenwolf 0 1992 WILEY-LISS, INC.

et al., '89b). Collectively, these results suggest that cells lying lateral to the neural plate provide the extrinsic forces underlying bending. Lateral ectoderm and/or endoderm seemingly could generate forces for bending through medially directed spreading (i.e., the expansion of such epithelial sheets toward the neural plate). This spreading could arise owing to changes in cell shape and position within the sheet, as well as to an increase in epithelial cell number and in the amount of extracellular matrix within the sheet. Additionally, lateral mesoderm presumably could generate forces for bending by "accumulating" beneath the lateral aspects of the neural plate. Such accumulation could result from the aggregation of cells migrating into this area from more lateral regions or to the proliferation and/or elongation of paraxial mesodermal cells underlying the neural plate. These events are characteristic features of somitogenesis, a process correlated both spatially and temporally with bending of the neural plate (Schroeder, '70; Lipton and Jacobson, '74). The purpose of this study was to examine the possible roles of the lateral surface epithelium, meso-

Received February 20,1991; revision accepted J u n e 6,1991. Address reprint requests to Dr. Gary C. Schoenwolf, Department of Anatomy, University of Utah, School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132.

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MATERIALS AND METHODS Chick embryos at stages 4-7 (Hamburger and Hamilton, '51) were cultured ventral-side up in New ('55) culture, modified as described previously (Schoenwolf et al., '89a). Mesoderm and endoderm were depleted in three locations (Fig. 1):the first, called lp (lateral neural plate), consisted of the area underlying the left lateral neural plate (47 embryos: 7 at stage 4, 12 at stage 5, 21 at stage 6, and 7 at stage 7); the second, called np (neural plate), consisted of the area underlying the left lateral neural plate as well as that underlying the midline neural plate (14 embryos: 11 at stage 5 and 3 at stage 6); and the third, called ps (primitive streak), consisted of the area lateral to the left side of the primitive streak (8 embryos: 3 at stage 5 and 5 at stage 6). A sharpened tungsten needle was used to remove a large piece of endoderm at one of the loca-

tions in each embryo. Then, the loosely packed, ingressed mesodermal cells were scraped away with a dull tungsten needle. For location np, the notochord (head process) was also removed; this was done with a sharp tungsten needle, taking care not t o damage the closely attached neural plate. In about half the cases at each location, a drop of solution consisting of trypsin/EDTA in saline (Irvine scientific, Santa Ana, CA; stock solution diluted 1 : l O with 123mM NaC1) was added, immediately after removal of the endoderm, to the exposed mesoderm, facilitating its removal (cultures were incubated with trypsin/EDTA for 10 min, followed by exhaustive washing with saline and replacement of the original culture plates with fresh ones). All cultures were incubated at 38°C in humidified chambers for 12 h, after which most had reached stages 10-12. Controls were of two types. Type 1 controls consisted of 15cultures in which the endoderm was removed partially and a drop of trypsidEDTA was added for 20 min, followed by washing. Type 2 controls consisted of 15 cultures in which the endoderm and mesoderm were depleted in the three locations (five cultures for each location) and then collected after 0-2 h of incubation. All cultures were videotaped (and selected ones photographed) immediately after completion of the operation to provide permanent documentation of the extent of endoderm and mesoderm depletion. Each was again videotaped (and some photographed) when the experiment was terminated. Embryos were fixed with either a 1 2 7 mixture of glacial acetic acid, 37% formaldehyde, and absolute ethanol or a 9:l mixture of absolute ethanol and 37%formaldehyde,and they were then processed for paraffin sectioning. Serial sections at 5 p,m were

Fig. 1. Schematics of ventral views of chick blastoderms showing the locations at which endodermal and mesodermal tissues were removed. lp, removal oftissues underlying the left (apparent right in ventral view) lateral neural plate; np, removal

of tissues underlying the left lateral neural plate plus those underlying the midline of the neural plate; ps, removal of tissues underlying the left side of the epiblast immediately lateral to the primitive streak.

derm, and endoderm in bending of the neural plate. To do this, endoderm and mesoderm underlying various regions of the epiblast were removed microsurgically at early stages of neurulation, and the effect of such removal on formation of the neural tube was examined; for technical reasons, the complementary experiment of removing only the surface epithelium was not done. Our results lead to the conclusion that the major extrinsic force for neurulation is generated by the surface epithelium, because bending of the neural plate still occurs after removal of the two other non-neuroepithelial tissues, but not of all three as reported previously (Schoenwolf, '88). Furthermore, we conclude that mesoderm (and perhaps endoderm) stabilizes the neuraxis, maintaining its proper orientation and position on the midline, because the neuraxis rotates toward the depleted side.

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stained either accordingto the Feulgen-Rossenbeck technique (asdescribed by Lillie, ’65)with fast-green counterstaining (embryos fixed with acetic acid, formaldehyde, and ethanol) or with Alcian blue (at pH 2.5, as described by Humason, ’72)with safTranin 0 counterstaining (embryosfixed with ethanol and formaldehyde).

RESULTS Controls Type 1controls developed normally and completed neurulation during the period of culture. Embryos were similar, in both their morphology and rate of development, to other control cultures (i.e., not treated with trypsin/EDTA) observed in our laboratory over the last several years. No difference could be detected in the amount of extracellular matrix present in these embryos (as revealed by staining with Alcian blue) compared with sections from untreated embryos. Sections of type 2 controls revealed the extent of tissue removal at the time of operation. At the operation site, virtually all endoderm and mesoderm was removed routinely, as well as much of the associated extracellular matrix (Fig. 2). The amount of extracellular matrix remaining at and neighboring the operation site appeared similar, regardless of whether embryos were treated with trypsinl EDTA. Furthermore, the efficacy of tissue removal was not stage or location dependent; in all cases, sections revealed that the objective of the operation was achieved.

Fig. 2. Transverse sections. A Section stained according

to the Feulgen-Rossenbeck procedure showing the effects of tissue removal at location np (embryo collected 1h later). B: Section stained with Alcian bluelsaffranin showing the effects of tissue removal at location lp (embryo collected 0.5 h later); note the depletion of the endoderm and mesoderm at the extirpation site as well as the depletion of the neural plate basement membrane (absence of dark staining, as is present bilateral to the extirpation site). e, endoderm; m, mesoderm; np, neural plate; arrows, extent of tissue removal. A, x 130; B, x 420.

Experimen tals Depletion of endoderm and mesoderm at location lp Neurulation occurred similarly throughout this group, irrespective of the stage at which the operation was performed, although some other aspects of development were affected in varying degrees depending on the stage of the embryo at the time of operation. Ventral views of cultures at 0 and 12 h following tissue removal at location lp are shown in Figure 3. Note the precision of the extirpations (Fig. 3A,C) and the subsequent development of the embryos, which was basically normal (Fig. 3B,D). Neurulation seemed t o be relatively unaffected by extirpation: the neural plate underwent virtually normal shaping and bending, and closure of the neural groove occurred throughout most (or all) its length. Nevertheless,two anomalies of the neural tube were noted in most embryos. First,the rostral portion of

the neural tube was rotated toward the side of tissue depletion. Second, localized dysraphism (see description of sections below) was present frequently. In addition to these relatively subtle effects on neurulation, extirpation often inhibited formation of the heart on the left side, but a heart tube still formed on the contralateral side; in embryos reaching advanced stages, this “half heart” beat regularly. Extirpation also inhibited the formation of other mesodermal rudiments, including the somites. When the extirpation was done at the earlier stages (i.e., stages 4 and 5),only the most rostral somite(s) was absent on the left side; progressively more were lacking as the operation was performed at advancing stages (i.e., stages 6 and 7). Sections revealed that the forebrain was malformed in most embryos, presumably owing to mechanical constraints. At the midbrain through spinal cord levels, the neuraxis had a remarkably normal morphology, despite the fact that most of

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Fig. 4. Transverse sections stained according to the FeulgenRossenbeck procedure from embryos collected 12 h after tissue removal at location lp. A-C: Tissue removed at stages 6,6,and 7, respectively (A and B are from two levels of the same embryo). Note that the spinal neurocele (C) exhibits occlusion h e . , the apical sides of its lateral walls are closely apposed). mb, midbrain; hb/sc, caudal hindbraidcranial spinal cord; arrowheads, neural crest cells; arrows, extent of tissue removal; diagonal line, orientation of the dorsoventral axis of the neural tube. x 140.

the mesoderm and endoderm were absent on the left side (Fig. 4). In all cases, the neural fold on the operated side, like the neural fold on the intact side, Fig. 3. Whole blastoderms viewed from the ventral side show- underwent complete or almost complete elevation ing the effects of tissue removal at location lp. A Stage 5 embryo toward the dorsal midline. Whether the twofolds at time of tissue removal; B: 12 h later. C: Stage-7 embryo at also came into contact and underwent fusion was time of tissue removal; D: 12 h later. Arrows, extent of tissue highly variable; consequently,Some dysraphism, as removal; arrowheads, level of rotation of the neural tube toward mentioned above, was common (Fig. 4B).This varithe side of tissue depletion. A, x 30; B-D, x 20.

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ation occurred among different embryos operated on at the same stage and within different levels of the same embryo, and it occurred irrespectively of whether embryos were treated with trypsidEDTA. Frequently, the spinal neurocele exhibited occlusion, a process that begins shortly after fusion of the neural folds (Schoenwolf and Desmond, '84a,b; Desmond and Schoenwolf, '85)and presumably plays a role in rapid brain enlargement, an event driven partially by neural tube fluid pressure (Desmond and Jacobson, '77). Also, at levels showing neural crest migration, neural crest cells accumulated beneath the neural fold on the operated, left side as a tight clump, rather than dispersing as on the unoperated, right side. In many cases, the formation and initial invagination of the auditory placodes occurred normally on both the intact and extirpated sides of the embryo, even though the underlying mesodermal and endodermal tissues were absent on the left side at this level. Sometimes the placode on the left side was smaller than that on the intact side, and in all embryos in which the operation was performed at later stages (i.e., stages 6 and 7) the left placode failed to form. The reason for this unexpected result is unknown. Depletion of endoderm and mesoderm at location np Ventral views of cultures at 0 and 12 h following tissue removal at location np are shown in Figure 5A,B. Embryos in this group had a gross appearance that was identical to that of embryos of the previous group. Most germane to the focus of this study, the rostra1 neural tube was rotated toward the site of tissue depletion and localized dysraphism was common, as for the previous group. Transverse sections of embryos collected immediately after tissue extirpation revealed that in all cases both axial and paraxial mesoderm and associated endoderm were depleted. Such depletion was still evident after 12 h of further development (Fig. 5C,D). The notochord and adjacent paraxial mesoderm were absent typically throughout the length of the midbrain and hindbrain levels, and in some

Fig. 5. Whole blastoderms (A,B) viewed from the ventral side showing the effects of tissue removal at location np (stage 5 embryo at time of tissue removal, A; embryo a t 12 h later, B), and transverse sections (C,D) stained according to the FeulgenRossenbeck procedure (embryo collected 12 h after tissue removal a t location np).mb, midbrain; hb/sc, caudal hindbraidcranial spinal cord; arrowheads, neural crest cells; arrows, extent of tissue removal. A, x 30; B, x 20; C,D, x 140.

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cases at the cranial spinal cord level as well. In the absence of a notochord for the 12 h period following its extirpation, the cells of the midline neural plate (median hinge point or MHP cells) lost some of their characteristic morphological features. At caudal levels (i.e. caudal hindbraidcranial spinal cord levels; Fig. 5D), midline cells appeared identical to more lateral cells (i.e., they were 13 as tall as lateral cells, rather than being short like normal MHP cells; and 21 spindle shaped, like more lateral cells, rather than being wedge shaped like normal MHP cells), but more cranially (midbrain and cranial hindbrain) they retained some of their features, although these features were not as pronounced as in control embryos with intact notochords. At caudal levels, occlusion of the spinal neurocele failed to occur. This was presumably owing to the altered shape of the cross-sectional profile of the spinal cord, namely, with its more circular rather than slitlike lumen. Depletion of endoderm and mesoderm at location ps Ventral views of cultures at 0 and 12 h following tissue removal at location ps are shown in Figure 6A,B. Other than exhibiting a marked curvature of the neuraxis and a deficiency of lateral tissues at the level of the extirpation site, these embryos appeared normal. Transverse sections revealed that the intermediate mesoderm and lateral plate mesoderm were absent throughout large extents of the spinal cord level, yet normal neurulation, with closure of the neural groove, still occurred (Fig. 6C). Furthermore, the spinal neurocele exhibited typical occlusion.

DISCUSSION The results of the present study suggest that extrinsic neurulation forces are generated by the surface epithelium lying lateral to the neural folds and further show that such forces are sufficient to effect neural plate bending. In the absence of mesoderm and endoderm (i.e., extirpation at locations lp, np, or ps), the neural plate undergoes normal shaping and substantial bending. During shaping, the neural plate on the average thickens apicobasally, narrows transversely, and lengthens longitudinally (Burnside and Jacobson, '68; Jacobson and Gordon, '76; Morriss-Kay, '81; Jacobson and Tam, '82; Schoenwolf, '85;Tuckett and Morriss-Kay,'85). Shaping occurs as a result of intrinsic forces (Schoenwolf, '88), and it involves changes in neuroepithelial cell shape, oriented cell division, and cell rearrangement (summarizedby Schoenwolf and

Fig. 6. Whole blastoderms (A,B) viewed from the ventral side showing the effects of tissue removal a t location ps (stage 6 embryo at time of tissue removal, A; embryo a t 12 h later, B), and a transverse section (C) stained according to the FeulgenRossenbeck procedure (embryocollected 12 h aRer tissue removal at location ps). s , somite; sc, spinal cord; arrows, extent of tissue removal; diagonal line, orientation of the dorsoventral axis of the neural tube. A, x 30; B, x 20; C, x 140.

Alvarez, '89). The fact that shaping still occurs in the absence of underlying tissues, as shown in the present study, suggests both that an underlying cellular substrate is not required for shaping and that shaping is independent of the mesodermal convergent-extension movements. This nonobligatory role in shaping for tissues underlying the neural plate was not clear from our previous study in which all three tissues lateral to the neural plate were extirpated (Schoenwolf,'88),because in

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that experiment mesoderm and endoderm already underlying the neural plate at the time of extirpation were left intact. Bending consists of two processes: furrowing and folding (reviewed by Schoenwolf and Smith, '90b). Furrowing of the neural plate, the formation of shallow, longitudinal grooves lined by wedgeshaped neuroepithelial cells, is restricted to three hinge points, the single median hinge point (MHP)and the paired dorsolateral hinge points (DLHPs); the later form principally at brain levels, whereas the former form throughout most brain and spinal cord levels. Folding of the neural plate occurs around the three hinge points and involves the elevation of the neural folds dorsally, with the axis of rotation centered at the MHP, and the convergence of the neural folds medially, with the axis of rotation on each side centered at the corresponding DLHP. In the absence of more lateral endoderm and mesoderm (extirpation at locations lp or ps),the MHP undergoes furrowing, and elevation of the neural folds occurs. In a previous study in which all tissues lateral to the neural plate were removed, the MHP still furrowed, but elevation of the neural folds failed to occur (Schoenwolf, '88). The previous study provides indirect evidence that mesoderm beneath the neural plate is not sufficient by itself (i.e., in the absence of more lateral tissues) to cause elevation of the neural folds. Prior to the present study, one possible way to interpret the previous result was that more lateral mesoderm, which was removed by the operation, normally spreads medially during neurulation to cause elevation. The present study shows that such spreading of the mesoderm is not necessary for neural fold elevation. Moreover, the present study demonstrates that if the midline endoderm and mesoderm are removed (extirpation at location np),furrowing of the MHP is inhibited but elevation of the neural folds still occurs. Finally, regardless of the type of operation, whether furrowing of the DLHPs, convergence of the neural folds, and fusion of the neural folds occurred was variable. How does the surface epithelium generate extrinsic neurulation forces? We showed in another study that surfkce epithelial cells undergo striking changes in behavior during neurulation, which would cause the epithelium to undergo expansion (Schoenwolf and Alvarez, '91); namely, surface epithelial cells 1)divide, increasing in number and increasing the overall volume of the surface epithelium; 2) change their shape by flattening, thereby increasing their surface area; and 3) rearrange, so that they move medially and caudally, streaming from the lateral regions of the area pellucida toward the neural folds.

Two other events could also contribute to expansion of the surface epithelium. 1)Development of the surface epithelium in early embryos is characterized by a progressive increase in the amount of intraepithelial and subepithelial hyaluronate (Solursh et al., '79); increase in the former would be expected to increase the volume of the epithelial sheet, leading to its expansion. In this regard, it is significant that chick embryos treated with hyaluronidase exhibit neural tube defects (Anderson and Meier, '82; Schoenwolf and Fisher, ,831,but whether this is owing specifically to depletion of the intraepithelial hyaluronate is unknown. 2) Radial intercalation of cells within the bilaminar surface epithelium (i.e.,this layer is composed of both a periderm and deep layer) might occur, thereby increasing its surface area in a manner similar t o that reported for the spreading (i.e., epiboly) of the animal cap ectoderm during gastrulation in amphibian embryos (Keller, '75, '76, '78, '80; Keller and Schoenwolf, '77). Finally, the surface ectoderm could play additional roles in neurulation during the formation of the neural folds. Two possibilities have been suggested: 1) that the surface ectoderm (and neural ectoderm) delaminates to produce the neural folds (MartinsGreen, '88) and 2) that the surface ectoderm serves as a substrate for "tractoring" of neuroepithelial cells during formation of the neural folds (Jacobson et al., '86; Moury and Jacobson, '89) (also see Schoenwolf and Smith, '90a, for discussion of these two possibilities). What roles do the mesoderm and endoderm play in neurulation? The presence of these tissues seemingly facilitates neurulation. In their absence, the neuraxis typically rotates toward the side of tissue depletion (reported previously by Meade, '821, and localized dysraphism is common. This suggests that the mesoderm (and perhaps endoderm) stabilizes the neuraxis by maintaining the latter's normal dorsal-ventral orientation and midline position. Presumably, this stabilization would facilitate neural fold apposition and fusion, increasing the likelihood that these processes would occur normally. Additionally, the presence of normal tissue relationships is required to generate a neuraxis with its characteristic shape. For example, the typical crosssectional morphology of the neural tube, with its thin floor plate and thick lateral walls, is a direct consequence of inductive interactions between the neuroepithelium and associated mesoderm (Holtfreter and Hamburger, '55; Watterson et al., '55; Weiss, '55; Grabowski, '56; Limborgh, '56; Malacinski and Youn, '81, '82; Youn and Malacinski, '81;

MICROSURGICAL ANALYSIS OF EXTRINSIC FORCES

Straaten et al., '88; Smith and Schoenwolf, '89; Placzek et al., '90). Additionally, the mesoderm likely acts as a scaffold. Although intimate spatial relationships between the neuroepithelium and mesoderm might be expected to have a role occlusion of the spinal neurocele (e.g., see Schoenwolf and Desmond, '84b: Fig. 23), the results of the present study, as well as previous results on the extirpation of somites shortly after neurulation is completed (Desmond and Schoenwolf, '86), argue against a role for paraxial mesoderm in this process (see Desmond and Field, '91, for other studies relevant to the initiation of occlusion). Moreover, the present experiments suggest that the notochord has only an indirect role in occlusion, that is, by inducing floor plate and, consequently, by the subsequent effect of the floor plate in establishing the cross-sectional, slitlike profile of the neurocele. In short, the results of the present study provide evidence that the major extrinsic force for bending of the neural plate is generated by the surface epithelium. Further studies on this tissue will be required to delineate precisely how cell behavior produces force.

ACKNOWLEDGMENTS The assistance of Theresa Oliver, Jennifer Parsons, and Fahima Rahman is greatly appreciated. I thank Dr. Mary Desmond for discussing with me unpublished research from her laboratory relevant to the present study. This research was supported by NIH grantNS18112 andby Fulbright Fellowship FU89-8797464. LITERATURE CITED Anderson,C.B., and S. Meier (1982)Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo. J . Exp. Zool., 221:329-335. Brun, R.B., and J.A. Garson (1983) Neurulation in the Mexican salamander (Ambystoma mexicanurn): A drug study and cell shape analysis of the epidermis and the neural plate. J. Embryol. Exp. Morphol., 74.275-295. Burnside, M.B., and A.G. Jacobson (1968) Analysis ofmorphogenetic movements in the neural plate of the newt Turicha torosa. Dev. Biol., 18:537-552. Desmond, M.E., and M.C. Field (1991) Evaluation of neural fold fusion and coincident initiation of spinal cord occlusion in the chick embryo. Submitted. Desmond, M.E., and A.G. Jacobson (1977) Embryonic brain enlargement requires cerebrospinal fluid pressure. Dev. Biol., 57:188-198. Desmond, M.E., and G.C. Schoenwolf (1985) Timing and positioning of occlusion of the spinal neurocele in the chick embryo. J. Comp. Neurol., 235:479-487. Desmond, M.E., and G.C. Schoenwolf (1986) Evaluation ofthe roles of intrinsic and extrinsic factors in occlusion of the spinal neurocoel during rapid brain enlargement in the chick embryo. J. Embryol. Exp. Morphol., 97:25-46.

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Expansion of surface epithelium provides the major extrinsic force for bending of the neural plate.

Neurulation, formation of the neural tube, requires both intrinsic forces (i.e., those generated within the neural plate) and extrinsic forces (i.e., ...
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